Sarf057 Development Of Improved Management Strategies For Red

Transcription

Sarf057
Development Of Improved Management Strategies For Red
Mark Syndrome (Rms)
A REPORT COMMISSIONED BY SARF
AND PREPARED BY
Professor Sandra Adams
Published by the: Scottish Aquaculture Research Forum (SARF)
This report is available at: http://www.sarf.org.uk
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This publication may be re-used free of charge in any format or medium. It may only be reused
accurately and not in a misleading context. For material must be acknowledged as SARF copyright
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Disclaimer
The opinions expressed in this report do not necessarily reflect the views of SARF and SARF
is not liable for the accuracy of the information provided or responsible for any use of the content.
Suggested Citation
Title: Development of improved management strategies for Red Mark Syndrome (RMS)
ISBN: 978-1-907266-49-2
First published: June 2012
© SARF 2010
Project Final Report Form
Please complete this form including an Executive Summary and the Final project report and return
by email to: [email protected]
SARF, PO Box 16, Birnam, Dunkeld, Perthshire PH8 0WU, Scotland
Project Details
SARF Project ID Code: SARF057
Project Title: Development of improved management strategies for Red Mark Syndrome (RMS).
Project: Start date 1st February 2010
End date 1st August 2011
Name(s) and address(s) of contractor organisation(s): Institute of Aquaculture, University of Stirling
Contractor’s Project Manager: Professor Sandra Adams
SARF Project Manager: Mr Richard Slaski
Total SARF Project costs £ £60,000
Total approved project expenditure £142,717
Total actual project expenditure £166917
Total *approved staff input 1.24 yrs
Total *actual staff input £1.57 yrs
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question requires a YES/NO answer only. All other details of any Intellectual Property must be included
under the Scientific Report or in an accompanying Annex).
.........YES NO
*Staff years of direct science effort
NOTES
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Scientific objectives
List the scientific objectives as set out in the contract. If necessary these can be expressed in abbreviated
form. Indicate where amendments have been agreed with the SARF Project Manager, giving the date of
amendment.
The original objectives were:
1. Collect samples
2. Characterisation of F. psychrophilum from clinical samples
3. Determine if the host fish develop antibodies to F. psychrophilum or/and RLO during an RMS
infection
4. Identification of additional RLO genes in RMS and SD samples.
5. Development of an archive qPCR for the RLO agent
6. Ring trial of samples for presence of RLO agent using conventional PCR
7. Investigate if the application of novel biological anti-bacterial treatment methods can resolve RMS
lesions
8. Development of management strategies.
Objectives 4 and 7 were amended at annual meetings, as follows:
 It was agreed that that Objective 4 was no longer required as an effective qPCR had been
developed in Objective 5.
 It was agreed that Objective 7 would be replaced with “Test the efficacy of an existing commercial
SRS vaccine against RMS” to determine of cross protection against RMS could be achieved.
Milestones
List the milestones. It is the responsibility of the contractor to check fully that all milestones have been
met and to provide a detailed explanation if this has not proved possible.
Milestone
Number
1
2
3
4
Target Date
30/9/2010
21/10/2010
Milestone Met
In Full
On
Time
Yes
Yes
Yes
No
31/12/2010
Yes
Yes
31/3/2011
NA
NA
Title
Collection of samples (serum and tissues
Characterisation of F. psychrophilum from
clinical samples (serotyping using the new
Stirling serotyping system and PFGE analysis
Completion of ELISAs to determine if the host
fish develop antibodies to RLO or/and F.
psychrophilum during an RMS infection
Identification of additional RLO genes in RMS
5
6
7
8
9
and SD samples.
Development of an archive qPCR for the RLO
agent
Ring trial of samples for presence of RLO agent
using conventional PCR
Testing the efficacy of an existing commercial
SRS vaccine against RMS
Development management strategies
Final Report
31/3/2011
Yes
Yes
30/07/2011
Yes
No
30/07/2011
NA
NA
1/8/2011
1/8/2011
tbc
Yes
tbc
No
If any milestones have not been met please give an explanation below.
All of the objectives were met with the exception of Objectives 7 & 8. Unfortunately the vaccine that had
been provied was not a full commercial vaccine and therefore we were advised by the VMD not to proceed.
We intend to proceed with both the original Objective 7 and testing an SRS vaccine in an extension to this
project if possible. MSD Animal Health has indicated that they could provide such a vaccine. Despite
additional effort in staff time and reagents during this project some of the work remains to be completed due
the huge volume of data generated. We would like to request an extension to the project in order to
complete all the analysis and perform the addition tasks that will inform on management strategies. Our
colleagues at Marine Scotland have indicated that they would be prepared to join the project (‘in kind’
contribution) and assist on some of the tasks. It is therefore hoped that Milestones 7 and 8 can be taken
forward and completed in an extension to this project.
Declaration
I declare that the information I have given in this form and in any associated documentation is correct to the
best of my knowledge and belief.
Name: Alexandra Adams
Position held: Professor
Date: 30th November 2011
Executive Summary
The executive summary must not exceed 2 sides in total of A4 (minimum font size 10) and should be understandable to
the intelligent non-specialist. It should cover the main objectives, methods and research results, together with any other
significant events and options for new work (the box below will expand to accommodate the Summary).
Executive Summary
Red Mark Syndrome (RMS) first seen in 2003 1 is an emerging disease in the UK and the same, or very similar
conditions, may also be causing significant problems in Europe and USA. RMS quickly spread to over 50% of rainbow
trout (Oncorhynchus mykiss) farms2 and the presence of lesions contributes to the downgrading in the market value of
the fish and results in significant economic losses3. In order to inform on management strategies basic information on
the aetioogical agent was required. The aim of this project was to elucidate the role of F. psychrophilum and
Rickettsia-like organisms (RLOs) in Red Mark Syndrome (RMS) and to develop improved management
strategies.
The original objectives were:
1. Collect samples
3. Determine if the host fish develop antibodies to F. psychrophilum or/and RLO during an RMS infection
7. Investigate if the application of novel biological anti-bacterial treatment methods can resolve RMS lesions
Objectives 4 and 7 were amended at annual meetings, as follows:
 It was agreed that that Objective 4 was no longer required as an effective qPCR had been developed in
Objective 5.
 It was agreed that Objective 7 would be replaced with “Test the efficacy of an existing commercial SRS
vaccine against RMS” to determine of cross protection against RMS could be achieved.
All of the objectives were met with the exception of Objectives 7 & 8. Unfortunately the vaccine that had been provied
was not a full commercial vaccine and therefore we were advised by the VMD not to proceed. We intend to proceed
with both the original Objective 7 and testing an SRS vaccine in an extension to this project if possible. MSD Animal
Health has indicated that they could provide such a vaccine. Despite additional effort in staff time and reagents during
this project some of the work remains to be completed due the huge volume of data generated. We would like to request
an extension to the project in order to complete all the analysis and perform the addition tasks that will inform on
management strategies (Milestone 8).
The results of the project have provided an invaluable resource for future work on RMS as well informing on
management strategies for the disease. The roles of F. psychrophilum and Rickettsia-like organisms (RLOs) in Red
Mark Syndrome (RMS) were elucidated using a variety of serological and molecular technologies.
The role of Flavobacterium. psychrophilum in RMS
From the RMS sampling programme, 108 Gram -ve filamentous yellow pigmented bacteria were collected from both
healthy and RMS affected fish. Although the Flavobacterium spp. in this collection have not yet been fully speciated,
there does not appear to be a correlation between RMS-affected fish and the number of Flavobacterium spp. found
compared with RMS-free fish. Serotyping of the Flavobacterium spp. by Western Blotting resulted in many more
profile types than the original four found when the system was set up for Flavobacterium psychrophilum indicating that
species other than F. psychrophilum were present. Molecular analysis by polymerase chain reaction (PCR) to detect F.
psychrophilum (in an MSc project) initially appeared to contradict this finding until it was found that the PCR reacted
with Flavobacterium sp. closely related to F. psychrophilum, suggesting that the PCR was in fact not completely
specific for F. psychrophilum. Sequencing of the isolates needs to be completed to confirm this.
The use of immunohistochemistry (IHC) to detect F. psychrophilum in fixed tissue sections (internal organs) gave
negative results, although inconclusive staining was observed in a few sections from both from RMS-positive and
negative farms. In addition, there was no difference in the serology (determined by enzyme linked immunosorbent
assay, ELISA) between fish serum collected from fish on RMS-infected and non-infected farms. This was carried out to
determine if the RMS-affected fish developed antibodies against F. psychrophilum or/and RLO during an RMS
infection.
The role of Rickettsia-like organisms (RLOs) RMS
It was possible to identify an RLO in RMS and Strawberry Disease (SD, from the USA) samples using a qPCR method
with high numbers of positive samples found in affected fish. The amount of RLO in the RMS lesion was significantly
higher than the other organs or unaffected skin. However, attempts to detect the RLO in formalin-fixed wax-embeded
archive samples were unsuccessful, possible due to damaged DNA upon its extraction.
No conclusive results were obtained from the cell culture work performed during the study (in an attempt to isolate the
RLO), and although a variety of bacterial isolates were obtained on PsA, these remain to be identified (collected from
local fish farms). Several swabs from skin lesions and spleen of RMS-postive fish produced very small white colonies,
but it has not been possible to passage these cultures on to fresh medium.
Final conclusions, recommended management strategies and future work
In conclusion, although Flavobacterium species were found in fish (mainly on the skin and not in the internal organs)
with RMS, no firm association could be made between this bacterium and RMS. There was, however, a strong
association between presence of the RLO (in the lesion and internal organs) by qPCR and RMS-affected fish samples.
Although this does not prove causality of the disease by the RLO it is possible to say that that the RLO does appear to
be involved in the disease.
Further work clearly needs to be done before definitive management strategies can be recommended. With the strong
correlation to the RLO it is feasible to suggest that a vaccine against the RLO would be a logical next step.
Unfortunatley as an RLO hs not been isolated it is not possible to develop a traditional inactivated whole cell vaccine,
therefore we must rely on other methods to mitigate the effects of the disease. If further sequence data can be obtained
on the RLO then perhaps a recombinant or DNA vaccine approach could be envisaged in the future.
We have also looked at other studies to inform on management strategies and made initial conclusions from these. For
example, a large scale epidemiological study was conducted by CEFAS and Marine Scotland using questionnaires and
results will be published shortly. Although RMS now affects 60% of all UK rainbow trout farms in the UK, this means
that 40% of farms are still free of the disease. It is possible for them to remain RMS-free, or even to clear the disease
with some effort. By far the highest risk of introducing RMS to a farm is by moving live fish. This is an essential
practice for any farm, but selecting an RMS-free source will limit the risk of infection dramatically. As recent research
has shown, having less than four suppliers greatly reduces the risk. The second main route of infection is the water
source. This may seem difficult to control, but having an influence over what is upstream of the site might help to
prevent disease outbreaks. This ties in with the strict biosecurity that should be carried out on the farm. Recent research
has also suggested that mechanical handling should be kept to a minimum. There are farms that have cleared the
disease and have had no reoccurrence: this was achieved by implementing all of the above and completely clearing,
fallowing and disinfecting ponds.
It is possible to treat fish with RMS. The easiest way is to leave the fish and let the lesions resolve naturally. If possible
fish can be hand-graded and fish with lesions selected and left to allow the lesions to heal. Stress has been reported to
have different effects on RMS infection: some farmers have noticed that after stressing the fish the lesions healed up
more quickly, whereas other farmers have found stress aggravates the situation. Another possibility is to fillet the fish,
but that is not always an option. Topical treatments with all sorts of disinfectants have been tried: results are mixed, but
they appear to make the lesions appear less severe rather the resolving them. Affected fish can be treated with a range
of antibiotics, florfenicol and oxytetracycline being the two most commonly used. However, they should only be used
as a last resort: antibiotic resistance and residues in the flesh are issues not to be taken lightly, and in any case the
disease will resolve itself in time.
Despite additional effort in staff time and reagents during this project some of the work remains to be completed due
the huge volume of data generated, and a number of new questions have been raised. We would like to request an
extension to the project in order to complete all the analysis and perform the addition tasks that will inform on
management strategies. Our colleagues at Marine Scotland have indicated that they would be prepared to join the
project ‘(in kind’ contribution) and assist on some of the tasks.
Project Report to SARF
As a guide this report should be no longer than 20 sides of A4. This report is to provide SARF with details of the outputs
of the research project for internal purposes; to meet the terms of the contract; and to allow SARF to publish details of
the outputs. This short report to SARF does not preclude contractors from also seeking to publish a full, formal scientific
report/paper in an appropriate scientific or other journal/publication.
The report to SARF should include:

the scientific objectives as set out in the contract;

the extent to which the objectives set out in the contract have been met;

details of methods used and the results obtained, including statistical analysis (if appropriate);

a discussion of the results and their reliability;

the main implications of the findings;

possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).
References to published material
This section should be used to record links (hypertext links where possible) or references to other published material
generated by, or relating to this project (the box below will expand).
1. Verner-Jeffreys, D. W., Algoet, M., Feist, S. W., Bateman, K., Peeler, E. J., & Branson, E. J. (2006).
Studies on red mark syndrome, Finfish News no. 1, pp. 19-22
2. Noguera, P. (2008). Red Mark Syndrome, Fish Farmer, vol. 31, no. 2, p. 38
3. Verner-Jeffreys, D. W., Pond, M. J., Peeler, E. J., Rimmer, G. S. E., Oidtmann, B., Way, K., Mewett,
J., Jeffrey, K., Bateman, K., Reese, R. A., & Feist, S. W. (2008). Emergence of cold water
strawberry disease of rainbow trout Oncorynchus mykiss in England and Wales: outbreak
investigations and transmission studies, Diseases of Aquatic Organisms, vol. 79, no. 3, pp. 207-21
4. Adam, K., 2009. A retrospective epidemiological study of red mark syndrome in Scottishfarmed
rainbow trout (Oncorhynchus mykiss). Marine Scotland – Science, Internal Report No 14/09.
SARF 057 Project Final Report
Development of Improved Management Strategies for Red Mark Syndrome (RMS)
A. Original Scientific Objectives
The aim of this project was to elucidate the role of F. psychrophilum and Rickettsia-like
organisms (RLOs) in RMS and to develop improved management strategies.
The original scientific objectives as set out in the contract were as follows:
1. Collect samples
3. Determine if the host fish develop antibodies to F. psychrophilum and/or RLO during an
RMS infection
7. Investigate if the application of novel biological anti-bacterial treatment methods can
resolve RMS lesions
Objectives 4 and 7 were amended at the review meetings, as follows:
 It was agreed that that Objective 4 was no longer required as an effective qPCR had
been developed in Objective 5.
 It was agreed that Objective 7 would be replaced with “Test the efficacy of an
existing commercial Salmon Rickettsial Syndrome (SRS) vaccine against RMS” to
determine if cross protection against RMS could be achieved.
B. Extent to which the objectives in the contract have been met
All of the objectives were met with the exception of Objective 7. Unfortunately the vaccine
that had been provided was not a full commercial vaccine and therefore we were advised by
the Veterinary Medicines Directorate (VMD) not to proceed. We intend to proceed with the
testing of an SRS vaccine in an extension to this project if possible. MSD Animal Health has
indicated that they could provide such a vaccine. Objective 8, development of management
strategies also requires to be completed once all the analysis of data has been finalised.
C. Methods, results and discussion for each objective
Objective 1 Collect samples (serum and tissues)
Approaches and Research Plan as outlined in the contract
Samples were to be collected by CEFAS and Stirling from the UK (RMS positive and
negative sites), Denmark (RMS negative samples by Dr Inger Dalsgaard and Lone Madsen
from DTU Aqua, Copenhagen) and the USA (SD positive samples) from Dr Douglas Call’s
group from the University of Washington.
Methods, results and discussion
Sample Set 1– RMS samples
Samples were collected in collaboration with the CEFAS Fish Health Inspectorate from two
RMS- positive and two RMS-negative sites in the UK (10 fish per farm). Bacterial swabs
were taken from the exterior and interior of lesions and mucus on the surface of ten fish at
1
each site, sampling these onto three different culture media; MV Agar (MVA) suitable for the
isolation of F. psychrophilum; Ps Agar (PsA) suitable for the isolation of Piscirickettsia
salmonis (used with the aim of isolating the RLO associated with RMS) and tryptone soya
agar (TSA). Tissue samples were also collected into 95 % ethanol from these fish for qPCR
analysis, as outlined in Objective 5, neutral buffered formalin for histopathology and for
immunohistochemisty (IHC) using anti-P.salmonis monoclonal antibodies (MAbs) and rabbit
polyclonal anti-F. psychrophilum serum. Sera were collected from these fish for screening
antibody responses against F. psychrophilum and P. salmonis in Objective 3. Further RMSnegative samples were obtained from the Netherlands (Mr Hendrik Hamstra), but originating
from Denmark (both countries have not been reported as having outbreaks of RMS). SDpositive samples were obtained from Dr Scott La Patra at Clear Spring Foods USA, and
together these samples were used as negative and positive controls in the IHC and the qPCR
analysis. Dr Douglas Call's group at the University of Washington performed the qPCR on
these samples following DNA extraction in the UK as part of the ring tesing performed in
Objective 6.
Tissues from the British farms were stained with H&E for histology, which were
examined and scored by Dr Steve Feist, the senior histopathologist at CEFAS. None of the
samples from the RMS-negative farms showed signs of pathology, except one fish which had
mild focal mysositism, but this was considered incidental and not related to RMS. Of the
samples taken from the RMS-affected farms, from the first farm three fish showed no signs of
RMS and the remainder showed mild to moderate pathology, while the ten fish from the
second farm all showed moderate to marked pathology (Figure 1).
a
b
A
c
d
Figure 1. Examples of positive histology (a) Red Mark Syndrome (UK) affected and (b)
Strawberry Disease (USA). Both show a full thickness dermatitis expanding into the muscle
(40 x magnification) and Immunohistochemistry (c) Kidney SD USA and (d) RMS skin
lesion using antibodies against Piscirickettsia salmonis. Both show positive staining
indicating the presence of antigens in common with P. salmonis.
In contrast to the positive staining by IHC using antibodies against Piscirickettsia salmonis
on RMS infected fish tissues (Figure 1d), when the tissues were using rabbit polyclonal antiF. psychrophilum antibody (PAb), the majority of the samples (diseased and normal skin,
2
kidney, liver, heart and gill) were negative. A few samples showed some slight, inconclusive
staining with the PAb.
Sample Set 2– Longitudinal RMS trial
Two duplicate longitudinal studies were performed in collaboration with Dr Scott La Patra at
Clear Spring Foods using specific pathogen free fish, with the aim of infecting them with
RMS through co-habitation infection. The design of this trial was in collaboration with Dr Ed
Peeler, an epidemiologist at CEFAS. Analyses by qPCR and IHC was performed on fish
sampled from the trials, the results of which are discussed under Objective 5.
Sample Set 3– Local Farms
Additional sampling was carried out at a local fish farm infected with RMS, to obtain
samples for cell culture in an attempt to isolate the RLO. Five fish with active lesions and
five with early lesions were sampled and their spleens placed on three different cell lines
(SHK-1, CHSE-214 and an insect cell line SF21 used to culture P. salmonis). Bacterial
cultures were also prepared to try to isolate RLO/ F. psychrophilum associated with RMS.
Blood from these fish was also cultured. No conclusive results were obtained from the cell
culture work, and although a variety of bacterial isolates were obtained on the P. salmonis
medium, these remain to be identified. Several swabs from skin lesions and spleen of RMSpostive fish produced very small white colonies, but it has not been possible to passage these
cultures on to fresh medium.
Sample Set 4–F. psychrophilum collection
As well as the clinical samples collected above, the F. psychrophilum collection at Stirling
was expanded (including Flavobacterium spp. from fish exhibiting signs of RTFS) and now
contains isolates from UK, Europe (France, Italy, Denmark, Spain, Finland), Japan, USA and
Chile. These include PFGE-reference isolates from Tim Wallis from Ridgeway Biologicals.
Currently 198 isolates have been collected.
Objective 2 Characterisation of F. psychrophilum from clinical samples (serotyping
using the new Stirling serotyping system and multigene analysis)
Isolates of F. psychrophilum isolated from RMS-affected fish were to be serotyped using
Stirling’s new newly developed serotyping system and the plan was also to analyse the
isolates by pulsed-field gel electrophoresis, and if necessary multilocus sequence typing.
Reference isolates and protocols were supplied by Dr Douglas Call, Dr Inger Dalsgaard and
Dr Tim Wallis from Ridgeway Biologicals. Dr Call has recently published a PFGE-based
typing scheme for F. psychrophilum isolates from the USA. Similarly, Dr Wallis has been
involved in a study that determined the clonal relatedness of isolates from UK rainbow trout
and Atlantic salmon. Dr Dalsgaard’s group has internationally recognized expertise on F.
psychrophilum and has also characterised isolates using a variety of methods. We wanted to
determine if there was a correlation between RMS-affected fish and distinct clades of F.
psychrophilum based on the subtyping data. CEFAS had experience with subtyping other
bacterial fish pathogens (e.g. Yersinia ruckeri) that could be used to help guide the project.
This objective was necessary so that a comparison could be made between F. psychrophilum
isolates collected from RTFS infected fish and those isolates found in association with RMS.
This information also assisted in selection of the F. psychrophilum isolates to be used to coat
the ELISA plates in Objective 3. The serotyping system was already set up and working at
Stirling.
3
Two bacterial collections were characterised i.e. the samples collected from the RMS-postive
and negative farms (the RMS collection) and also a large F. psychrophilum collection.
The RMS Collection
Swabs were made from mucus, interior and external skin lesions of RMS-affected (Farms 3
and 4) and healthy fish (Farms 1 and 2), and these were subcultured onto the three different
culture media mentioned above i.e. TSA, MVA and PsA. The aim of the latter was to try to
isolate a Rickettsia-like Organism (RLO) associated with RMS. The cultures on TSA and
PsA had a large amount of non-specific growth on them and were therefore dicarded, while
108 Gram -ve filamentous yellow pigment bacteria were collected from the MVA cultures,
distrubuted in tissues as shown in Table 1, and 52 of these were thought to be potential
Flavobacterium spp (13 from APIzym (Table 2) and 39 based on morphology, ELISA and
IFAT).
Table 1. Gram –ve filamentous yellow pigment bacteria were collected from the MVA
cultures cultures
Farm
Mucous
Exterior
Interior
1 (-ve)
(17)
(10)
(7)
2 (-ve)
(11)
(7)
(6)
3 (+ve)
(5)
(13)
(11)
4 (+ve)
(10)
(9)
(7)
Table 2. Potential Flavobacterium spp based on APIzyme
Farm
Mucous
Exterior
1 (-ve)
(2)
(0)
2 (-ve)
(1)
(4)
3 (+ve)
(1)
(2)
4 (+ve)
(0)
(0)
Interior
(0)
(1)
(1)
(1)
The RMS collection was kindly analyzed by a collaborator in Korea using a MALDI
BioTyper™ system. This method allows identification and classification of microorganisms
using protein 'fingerprints' measured by MALDI-TOF mass spectrometry, and it is becoming
a very popular method for analysing clinical isolates from human infections. Bacterial
identification is performed using pattern matching between reference spectra and MALDITOF profiles of unknown strains and it can identify bacteria to the genus or species level. The
method has the potential to differentiate between microbial strains and allows clustering and
phylogenetic dendrogram construction between isolates, similar to the PFGE (which is based
on DNA analysis rather than protein profiles).
Preliminary data is shown in the dendograms in Figure 2. Of the isolates classified as
Flavobacterium sp. many were classified as F. saccharophilum, however bacterial
characterisation is only as good as the reference isolates used and the RMS isolates were
compared with reference bacteria held in the software database used to characterise clinical
isolates from human infections.
4
MSP Dendrogram
R62
R147
R36
R78
R34-1
R35
ps M174
R138
Pseudomonas spp. cluster
R138 New
R132
PS M 162
R146
R141
R144
R140
R139
R143
unindentified spp.
Bacillus
R149
R86
R100
R59
R39
R97
R77
R98
R40
R126
R128
R129
Flavobacterium spp. cluster
R127
1000
900
800
700
600
500
Distance Level
400
300
200
100
0
I
MSP Dendrogram
1000
900
800
700
600
500
Distance Level
400
300
200
100
R33
R116
R91
R118
R80
R79
R21
R18
R17
R84
R111
R87
R24
R53
R4
R88
R89
R107
R136
R121
R112
R103
R106
R102
R5
R125
R3
R2
R57
R7
R83
R30
R58
R16
R14
R137
R135
R134
R108
R92
R90
R48
R120
R82
R81
R32
R115
R114
R15-1
R19
R20
R15
R105
R104
0
Flavobacterium sp cluster
Pseudomonas spp. cluster
Figure 2 MALDI BioTyper™ analysis for the RMS isolates
5
Approximately half of the isolates could not be identified as there were no related reference
stains in the database. The RMS isolates are currently being reanalysed using aquatic
reference type stains including more Flavobacterium sp. type stains (including four F.
psychrophilum type strains). At this stage there does not appear to be a correlation between
the isolates identified as Flavobacterium spp. (mainly classified as F. saccharophilum
according to the database) from the RMS-positive or RMS-negative farms, or the type of
sample taken (i.e. swabs from mucus or interior/exterior lesions), but additional analysis is
required to identify the isolates further. Perhaps a more accurate identity will be found for the
F. saccharophilum isolates. The relationship between the results of the MALDI BioTyper™
and the results obtained with the commercially available Mab against F. psychrophilum in
immunofluorescent antibody technique (FAT) and in ELISA using the polyclonal rabbit anti
F. psychrophilum sera still need to be evaluated. This constitutes a very large data set and we
propose to perform this over the next 6 months. Also DNA from all RMS isolates has been
extracted with a view to sequencing the 16S RNA of these bacteria for bacterial
identification, but this has not as yet been sequenced. It is important to do this to verify any
relationship between the isolates identified by the MALDI BioTyper™ and in the other tests.
The F. psychrophilum collection
There are currently 198 isolates in this collection, with isolates obtained from fish exhibiting
signs of RTFS, and isolates from UK, Europe (France, Italy, Denmark, Spain, Finland),
Japan, USA and Chile, as well as isolates from Atlantic salmon, Coho salmon and Rainbow
trout. The collection was first characterised based on bacterial morphology/Gram staining,
then serotyping, using the serotyping system developed at Stirling based on Western blotting
with polyclonal rabbit anti-F. psychrophilum sera (see Figure 3), FAT using the
commercially available Mab against F. psychrophilum and ELISA using the polyclonal rabbit
anti-F. psychrophilum sera.
Morphologically, there was great variation in colour, the swamping properties and the
colony types between the different isolates, but they were in general long filamentous Gram ve rods. In the FAT and ELISA with the rabbit PAb, most bacteria were positive to differing
degrees (+ - +++). When analysis was repeated using anti-F. psychrophilum Mabs
approximately one third of the F. psycrhophilum collection were recognised with the
commercial Mab, and one third with an in house F. psychrophilum MAb. A Mab against F.
psycrhophilum ECPs reacted with most of the isolates to differing degrees in ELISA (+ +++).
Serotyping
Originally the serotyping system developed at Stirling, based on Western blotting with
polyclonal rabbit anti-F. psychrophilum sera, identified four different serotypes characterised
by the banding patterns shown in Figure 3. However, when this method was applied to the
Flavobacterium collection isolates, many more profiles than the original four were obtained.
An example of some of the profiles obtained is shown in Figure 4. As yet it is not known
how these profiles relate to different Flavobacterium spp. isolates, therefore completing the
16S RNA sequencing outlined below is crucial. The polyclonal sera used are not completely
specific for F. psychrophilum, and is known to cross-react with F. branchopilium and closely
related F. psychrophilum spp. This may explain why so many profiles were obtained, as some
of these isolates are known not to be F. psychrophilum (see below).
6
KDa
160
75
50
35
30
25
15
10
A
B
C
D
Figure 3. Serotyping system developed at Stirling based on Western blotting with polyclonal
rabbit anti-F. psychrophilum sera
Figure 4. Examples of the Serotyping performed on the Flavobacterium sp. collection. The
numbers refer to different isolates.
MALDI BioTyper
The Flavobacterium collection was also analyzed using the MALDI BioTyper™, the results
of which, shown in the dendogram in Figure 5, show clear clusters between isolates. This is,
however, preliminary data since further Flavobacterium spp. type strains are required in the
database for more accurate characterisation of the collection. Also around a third of the
7
isolates were lost (deteriorated) on route to Korea because of a delay in their delivery by the
courier. The Flavobacterium isolates are currently being reanalysed using aquatic reference
type stains including four Flavobacterium sp. type stains.
MSP Dendrogram
F7
F39
F70
F33
F9
F61
F24
F8
F23
F19
F65
F64
F62
F40
F38
F29
F83
F48
F44
F43
F100
FC
F41
F82
F102
F36
F2
F107
F101
FA
F96
F56
F55
F25
F58
F30
F16
F69
F75
F74
F71
F6
F3
FJ
cert7357
F14
F17
F57
F35
F28
F12
F47
F46
F11
F18
FH
F45
1000
900
800
700
600
500
Distance Level
400
300
200
100
0
Figure 5. MALDI BioTyper™ analysis for the Flavobacterium collection
PCR and Sequencing
Some of the isolates have been analysed by a nested PCR (Wiklund et al., 2000), which was
apparently specific for F. psychrophilum. A number of the isolates from the collection
formed the basis of an MSc student project carried out in the Institute of Aqiaculture early
this year. It was found that the Flavobacterium sp. closely related to F. psychrophilum were
positive by PCR, suggesting that the PCR is in fact not completely specific for F.
psychrophilum. We need to complete the sequencing of the isolates to confirm this. DNA has
been extracted from the isolates ready for sequencing for species identification. This
information is also important to examine the relationship between the results of the MALDI
BioTyper™, IFAT and PCR (the PCR analysis also need to be completed on the remainder of
8
the isolates). A few of the isolates have already been sequenced and are not in fact F.
psychrophilum, but are closely related species.
PFGE
Dr Kim Thompson undertook two weeks of PFGE training with Dr Michelle Pond at CEFAS
in September 2011. Dr Pond has had experience with subtyping other bacterial fish pathogens
(e.g. Yersinia ruckeri) and this work together with published PFGE protocols for F.
psychrophilum helped in the optimisation of the technique for this project. The PFGE system
at Stirling is different to the one at CEFAS and the technique needed to be re-optimized for
the Stirling system. Completion of this task was rescheduled for the end of June 2011, but it
was decided that is was necessary to complete the speciation of the Flavobacterim collection
and the RMS samples first so that the groupings obtained with the PFGE results would have
some significance with respect to different Flavobacterium spp.
Antibiotic resistance
The Flavobacterium sp. collection was also used in another MSc project earlier this year
looking at the antibiotic resistance of the isolates, as a collaborative research project between
Institute of Aquaculture, CEFAS, and MSD Animal Health. Initial results are very interesting
and it would be informative to tie in the typing results with these data once further analysis
has been completed.
Objective 3 Determine if the host fish develop antibodies to F. psychrophilum or/and
RLO during an RMS infection
Detection of specific antibodies in the serum of animals can be a useful indicator of previous
exposure to pathogens and is regularly used in both clinical and veterinary medicine. It also
has potential use for disease surveillance in aquaculture. This type of serology is often used
when rapid tests to identify the pathogen have not yet been developed. Such methods are
presently under used in aquaculture although their potential in disease management in other
animals is well proven.
Serum samples (both positive and negative for RMS) were analysed by enzymelinked immunosorbent assay (ELISA) to determine the host response to F. psychrophilum
and RLOs (Stirling). The aim was to help elucidate the role that the two pathogens
(individually or together) may have in RMS and may also lead to useful methods for
diagnosing the disease. Serum samples (both positive and negative for RMS) were analysed
by ELISA to determine the host response to the two pathogens (i.e. the F. psychrophilum and
RLO) (Stirling).
Serum samples from both the RMS-positive and RMS-negative sites, sampled under
Objective 1, were analysed in an ELISA to determine the host response to F. psychrophilum
and RLOs and to help elucidate the role that these two pathogens in RMS. The sera were
screened against four isolates of Flavobacterium spp. (a F. psychrophilum type strain and
three closely related Flavobacterium sp.) and also P. salmonis. As the RLO associated with
RMS has still not been isolated, it was decided to use P. salmonis in this screening since
MAbs against P. salmonis react with RMS-infected tissue suggesting common antigens
between the RLO and P. salmonis, which the fish immune response may also recognise. No
statistical difference was found in the antibody responses between the bacteria species or
between RMS-postive and RMS-negative sites (Table 3). Antibody titres against the five
pathogens were generally very low and ranged between 1/64 and 1/256.
9
Table 3. Antibody titre (-Log2+1) of sera sampled from RMS positive and negative farms
NCIMB FpT ARF07
Mof25
BGARF
P. salmonis
Farm 1
5.8 ±1.4
8.1 ±1.2
7.0±1.2
7.9 ±0.7
7.7 ±0.8
Farm 2
7.5 ±1.0
7.1 ±0.6
7.6 ±1.2
7.3 ±0.5
7.4 ±0.5
Farm 3
7.3 ±2.4
6.9 ±2.4
7.1 ±2.3
6.9 ±2.4
7.0 ±2.4
Farm 4
8.0 ±0.5
7 .0±0.5
7.5 ±0.5
7 .0±0.5
7.8 ±0.4
Results represent mean of 10 duplicate samples per farm
Objective 4 Identification of additional RLO genes in RMS and SD samples
Expanding on the initial work done by Dr Call's group RMS- and SD-positive samples were
to be probed by PCR for the presence of RLO genes other then 16S rRNA gene sequences.
4.1. Identification of RLO genes in databases, alignment of candidate gene sequences and
design of degenerate primers.
4.2. Test samples using degenerate primers and sequencing of resultant products, qPCR
developed and optimised for RLO (at CEFAS) and F. psychrophilum (at Stirling). This
will enable quantification of the two pathogens and will be used in task 5 below.
4.1. The identification of RLO genes in databases, alignment of candidate gene sequences
and design of degenerate primers was no longer required as a TaqMan qPCR assay was
optimised in this study based on the assay published by Doug Call’s group in USA, as
discussed under Objective 5. Attempts to development a qPCR for F. psychrophilum was
carried out at Stirling by PhD Student Farah Manji using primers against 16S rDNA, but
these were found to cross-react with other Flavobacterium spp. (F. aquatile and F.
johnsonaie). Primer sets for different F. psychrophilum genes should be considered for
further qPCR development.
Objective 5 Development of an archive qPCR for the RLO agent
Subject to identifying other RLO genes in RMS and SD samples, the obtained sequences
were then to be used to develop a TaqMan qPCR assay that could be used to screen samples,
both ethanol preserved from recent RMS outbreaks and formalin fixed material from the
CEFAS Registry of Aquatic Pathology (RAP; http://www.aquaticpathology.co.uk/). A
retrospective analysis using qPCR would be conducted to detect the presence of RLO DNA
in archived samples that have been collected by CEFAS from diseased rainbow trout over the
last 30 years. The presence or absence of RLO DNA in these samples would provide valuable
further information on whether the RLO is a genuinely new emergent disease-causing
organism. The RA from Stirling was based in CEFAS for this work. It was proposed to use
TaqMan qPCR assays for the qPCR which CEFAS have significant experience in developing
for disease diagnostics purposes. Successful development of an archive qPCR for the RLO
agent requires that a very sensitive quantitative assay is developed. This was the most
technically difficult part of the project.
5.1. Develop a TaqMan qPCR assay based on sequences obtained from 4.2 and testing the
RLO qPCR on positive control samples
5.2 Limited testing of archive and clinical samples using RLO TaqMan qPCR assay.
5.3 Cohabitation trial
10
5.1 A Taqman qPCR assay, developed by the University of Washington, targeting 16S rDNA
was performed at the CEFAS laboratory. Optimisation of the assay was carried out by
Matthijs Metselaar a PhD student from Stirling and Richard Paley from CEFAS. The
standards for quantification of the RLO consisted of a plasmid containing the sequence for
which the primers encoded (16S rDNA). A 10-fold dilution series of plasmid in a background
of RMS-negative whole fish DNA was prepared to mimic conditions in the test samples. The
assay was used to screen skin lesions, unaffected skin, heart, liver, gill, kidney and spleen
samples from the RMS-positive and RMS-negative sites, sampled in Objective 1. DNA was
extracted from samples stored in ethanol using a Qiagen biorobot 8000 universal system®
and Qiagen EZ1 at CEFAS, which gave constant quantity and high quality DNA. All qPCR
reaction were carried out in triplicate. At all steps during the proces relavant controls were
used (for example negative DNA extractions/no template qPCR reactions). As seen in Tables
4-9, there is a clear, strong correlation between the RMS affected fish and the RLO. In only 6
out of 210 samples tested from the disease negative farms was a positive amplification
observed for RLO DNA. In all six cases this was a single replicate of three (indicated by the
red high-lighting in Table 4-6), indicating either dubious positive or RLO is present in very
small amounts in these samples. The number of samples with positive amplification in the
affected fish from RMS positive farms was very much higher. Some but by no means all of
the positive amplifications observed in the affected fish samples were also in a single reaction
of triplicates. What can also be seen from the data is that the amount of RLO in the lesion is
significantly higher than the other organs and the unaffected skin as seen in the boxplot in
Figure 6.
This observed strong association however does not prove causality. Only by isolation
of the bacterium and fulfilment of Koch’s postulates we can definitively prove causality.
However, given the large amount of samples tested and the consistent results obtained, we
suggest that the RLO is at least involved in the disease.
Table 4. Farm 1 RMS negative farm from UK
Fish
1
2
3
4
5
6
7
8
9
10
Diseased
skin
-
Normal
skin
0.17*
-
Heart
Liver
Gill
Kidney
Spleen
-
-
-
-
-
Table 5. Farm 2 RMS negative farm from UK
Fish
1
2
3
4
5
6
7
8
Diseased
skin
-
Normal
skin
-
Heart
Liver
Gill
Kidney
Spleen
-
-
3.35* x10-6
-
3.97* x10-7
-
-
11
9
10
-
-
-
-
-
-
-
Table 6. Farm 3 RMS negative farm from NL
Fish
1
2
3
4
5
6
7
8
9
10
Diseased
skin
-
Normal
skin
-
Heart
Liver
Gill
Kidney
Spleen
-
1.79* x10-6
5.94* x10-6
-
-
-
2.46* x10-6
-
-
Table 7. Farm 4 RMS positive farm from UK
Fish
1
2
3
4
5
6
7
8
9
10
Diseased
skin
3.29 x10-5
1.87 x10-3
1.58 x10-4
3.14 x10-5
6.56 x10-4
1.55 x10-4
5.53 x10-6
1.34* x10-5
Normal
skin
9.35 x10-6
6.98 x10-5
1.17 x10-5
3.41* x10-5
Heart
Liver
Gill
Kidney
Spleen
1.26* x10-6
4.62 x10-6
1.42 x10-6
9.61* x10-7
6.65 x10-6
3.59* x10-6
1.20 x10-6
7.22* x10-7
3.69 x10-4
6.26* x10-7
1.55* x10-6
1.96* x10-6
9.13 x10-7
1.52 x10-6
9.01 x10-7
1.21* x10-6
-
1.26* x10-6
1.78* x10-6
2.21 x10-6
1.42* x10-6
-
Table 8. Farm 5 RMS positive farm from UK
Fish
1
2
3
4
5
6
7
8
9
10
Diseased
skin
8.38 x10-4
0.68**
6.07 x10-7
1.37 x10-6
4.71 x10-4
1.22 x10-4
7.35 x10-6
1.64 x10-4
Normal
skin
2.51 x10-5
1.44 x10-4
5.96 x10-5
1.18* x10-5
6.26 x10-5
1.38 x10-5
Heart
Liver
Gill
Kidney
Spleen
5.03 x10-6
4.00* x10-6
8.40* x10-9
1.36* x10-6
2.55 x10-6
1.11* x10-6
2.75* x10-6
8.80* x10-7
5.20* x10-7
1.40 x10-4
1.52 x10-6
9.40 x10-7
3.26* x10-7
1.15* x10-6
1.24 x10-6
8.40 x10-7
5.07* x10-7
8.57 x10-6
9.38 x10-7
5.56 x10-9
3.15 x10-6
2.93* x10-7
3.68 x10-6
4.14* x10-7
1.30475
1.63**
1.79 x10-6
2.71 x10-6
4.60* x10-6
4.70* x10-3
Table 9. Farm 6 RMS/SD positive farm from the US
Fish
1
2
3
4
5
6
7
8
Diseased
skin
7.41 x10-4
3.93 x10-4
1.86**
1.06 x10-3
2.28 x10-4
3.83 x10-5
1.78 x10-4
1.67 x10-4
Normal
skin
1.81 x10-5
2.89 x10-5
3.3* x10-5
1.57* x10-5
4.51 x10-4
Heart
Liver
Gill
Kidney
Spleen
3.83* x10-5
4.55 x10-6
2.92* x10-6
5.25 x10-6
5.43* x10-6
1.76* x10-6
39.23**
3.93 x10-6
1.19 x10-5
7.86 x10-6
1.10 x10-5
1.9 x10-6
1.02 x10-6
3.69 x10-5
5.92 x10-6
1.49 x10-3
7.39 x10-6
1.55 x10-6
3.72 x10-6
1.17 x10-5
7.11 x10-5
12
9
10
4.69 x10-5
1.20 x10-4
1.25 x10-4
2.04* x10-4
1.23* x10-5
-
1.00* x10-5
-
3.22* x10-6
1.26 x10-5
2.09 x10-6
7.64 x10-6
*insignificant; one of three replicates amplified
** outlier; one out 3 replicates significantly higher
- no amplification of the RLO
Table 4-9. Results from qPCR assays performed on RMS-affected and nonaffected fish. Numbers given are RLO copy number normalised by IGF copy number to
correct for the amount of DNA initially added to the reaction. The red highlighted
results are those of which only one of 3 replicates gave positive amplification.
Figure 6. Boxplot of combined results of the 3 affected farms. RLO copy numbers are
significantly higher in the skin lesions (Normal skin was taken from diseased fish)
5.2 Limited testing of archive and clinical samples using RLO TaqMan qPCR assay.
It was intended to use this assay to screen samples preserved in ethanol from recent RMS
outbreaks and formalin fixed material from the CEFAS Registry of Aquatic Pathology (RAP;
http://www.aquaticpathology.co.uk/), and to conduct retrospective analysis to detect the
presence of RLO DNA in archived samples collected by CEFAS from diseased rainbow trout
over the last 30 years.
DNA was extracted from archive wax embedded samples at CEFAS using their extraction
protocol. In total 10 samples were identified from the archive material which were positive
for RMS by histopathology. Unfortunally, no amplification was seen with the qPCR in any of
these samples. DNA extracted from the positive control, which was recently embedded tissue
from a fish that tested positive in conventional PCR and IHC, was also not amplified after
extraction from the wax block. It is possible that the DNA was degraded as a result of the
13
extraction proccess or from the formalin fixation. Futher work is needed to optimse the DNA
extraction protocol from these samples.
5.3 Cohabitation trials
In a duplicate study 100 and 98 naïve fish were cohabitated with 26 and 30 fish respectively
obtained from an SD outbreak (14.5°C). Skin and kidney samples from five fish were taken
at weekly intervals over the course of the trial (10 and 9 weeks respectively),. DNA was
extracted from these and the RLO-specific qPCR, developed by Lloyd et. al. (2011), was
used to monitor the transmission of the RLO. In cohabitation trial 2 additional organs (gill,
liver spleen and heart) were also collected and fixed for histology and IHC (Metselaar et. al.,
2010).
Clinical signs of SD were clearly evident in the cohabitated naïve fish of the first trial, while
the second trial did not reveal any transmission. Three fish showed typical signs of SD at 4, 7
and 10 weeks. Two of these fish were also positive for the RLO by qPCR in lesion and
kidney samples; one was also positive in normal skin, spleen and gill samples. In addition to
this, 2 fish were positive for the RLO by qPCR in kidney samples at week 9 of the trial, but
did not show lesions however. Histology revealed considerable inflammation and spongiosis
in the gills of these 5 fish. The 2 fish sampled at week 9 also showed inflammation in the
liver with focal necrosis and haemorrhages. One of the fish also had necrosis and
haemorrhages in the dermis with fibrin deposits. The changes observed in these fish can also
be seen in SD and RMS however. No obvious reaction was found with the P. salmonis
antibodies in IHC.
Mortality was seen in both studies (up to 54 %). Additional testing of moribund fish in trial 1
revealed the presence of IHN virus and bacterial infections in some fish, but not all (3/6). No
consistent single specific pathogen was found. Trial 2 had a similar pattern of mortality and
co-infection.
As the number of fish presenting typical SD lesions was very low and co-infections were
detected, no firm conclusions could be made about the transmission of the RLO. The results
can, however, be used to speculate about the role of the RLO in a farm situation. It appears
that the RLO may be involved early in the infection. At week 6 and 9 the RLO was already
present when the lesions appeared. This, together with previous findings (Lloyd, 2010),
suggests an association, but does not prove causation of the RLO. However, the 2 fish
sampled at week 9 did not have any lesions, although the RLO was present in their kidneys.
As the histology of the other internal organs was consistent with RMS/SD, this suggests
that this is a primary rather than secondary infection. Further work is needed to confirm this
however. The fish sampled at week 4 did have a lesion, but did not have the RLO in any of its
organs. It is possible that, as only a small sample of the organs was collected and low
concentrations of the RLO were present, and that the RLO was missed.
Objective 6 Ring trial of samples for presence of RLO agent using conventional PCR
Samples of rainbow trout displaying clinical signs consistent with RMS and a range of
negative control material from fish that had not reportedly been exposed to the disease were
collected. This material was probed for the presence of the RLO by PCR as described by
Lloyd et al., (2011). This was proposed to be performed in collaboration with international
workers, both Dr Douglas Call’s group from Washington State University, who formulated
the RLO hypothesis, and Dr Inger Dalsgaard’s group from DTU Aqua in Denmark. Denmark
is reportedly free of RMS, so fish sourced from Danish farms are an ideal source of negative
14
control material. The plan was to test the same material using the same reagents by the three
different laboratories (e.g. a ring trial was undertaken).
Samples of fish from the US, infected with SD, were supplied by Scott LaPatra and stored in
95% ethanol. These were then couriered to CEFAS, DNA extracted and analysed by qPCR.
Results of this are discussed in Section 5.1. The extracted DNA was then send back to the lab
at Washington University and analysed using their published qPCR method, which uses the
same primers and probes, but different Mastermix and reaction conditions. Only kidney, and
affected and unaffected skin were analysed by the Washington group. The results of their
analysis are shown in Table 11, together with the results of the UK analysis. Although the US
lab did find RLOs in these samples (Table 10), it can be seen that not all the samples which
tested positive in the UK lab were positive in the US analysis (Table 11).
Table 10. Farm 6 RMS/SD positive farm from the US tested in the US and the UK Comparison of RLO copy numbers of the same samples in the 2 different labs
Sample
US
UK
6.2.1
7433.8
430.8
6.4.1 132479.9 1634.9
6.5.1
78978.0 470.8
6.7.1
17121.4 133.1
6.8.1
12865.9 214.7
6.3.2
8710.7
16.9
6.8.6
31283.9
92.1
Due to communication problems the original DNA samples were diluted 1:10 compared to
the UK samples. This is the most likely cause why not all the samples, which tested positive
in the UK lab came up positive in the US lab (Table 11). However, if the quantitative data is
examined it can be seen that the US group found higher levels of RLO in the samples. This
was also apparent in the original US publication and the reason for this is unclear. The raw
data in Table 12, below, shows no statistical difference (student t test) but also no correlation
between the two sets of samples. We are still trying to establish the cause of this discrepancy,
but the most likely reason is the fact that different reagents, cycling conditions and the
standards were used by the two laboratories. Also the qPCR cycler machine on which the
assay is run can influence the reaction as we experienced when we tried to run the same
samples analysed at CEFAS on the thermo cycler at the Institute of Aquaculture.
Table 11 Results of qPCR for RLO performed in the US.
Fish
1
2
US
Diseased Normal
skin
skin
--+
--
UK
Kidney Diseased Normal
skin
skin
+
+
-+
--
Kidney
+
15
3
4
5
6
7
8
9
10
-+
+
-+
+
+/---
+
--------
+/-----+
---
+
+
+
+
-
-
+
-
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
Table 12 Comparison of normalised raw data
UK
US
Sample
RLO Ct/IGF Ct
Sample
RLO Ct/IGF Ct
6.2.1
6.4.1
6.5.1
6.7.1
6.8.1
6.3.2
6.8.6
1.621409
1.506375
1.684048
1.568689
1.62481
1.465831
1.627081
6.2.1
6.4.1
6.5.1
6.7.1
6.8.1
6.3.2
6.8.6
1.52993
1.68755
1.72145
1.58931
1.46974
1.62952
1.65668
Objective 7 Investigate if the application of novel biological anti-bacterial treatment
methods can resolve RMS lesions was amended to “Test the efficacy of an existing
commercial SRS vaccine against RMS”(amended following discussions at the 6 month
review meeting).
Results for amendment
Vetreqimica, a Chilean vaccine company provided a SRS vaccine for field trials to determine
if it would protect against RMS. We were, however, unable to obtain VMD approval to do
this in the field as it was unregistered vaccine.
Objective 8 Recommend Management Strategies
Information from this project, and associated projects that Stirling and CEFAS are involved
with, were to be used to recommend improved management strategies for RMS.
With the strong correlation to the RLO it is feasible to suggest that a vaccine against the RLO
would be a logical next step. Again we encounter the problem that the RLO has not yet been
isolated. Until this has been achieved it is not possible to develop a traditional inactivated
whole cell vaccine, therefore we must rely on other methods to mitigate the effects of the
disease. If future sequence data can be obtained on the RLO then perhaps a recombinant or
DNA vaccine approach could be envisaged in the future.
We have also looked at other studies to inform on management strategies and made initial
conclusions from these. For example, a large scale epidemiological study was conducted by
16
CEFAS and Marine Scotland using questionnaires. Data from Scotland has already been
distributed to the participating farms and data presented in flyers is available from both
institutes. Further data from this work is still being analysed and will be published shortly.
Prevention
Prevention is better than cure. Although RMS now affects 60% of all UK rainbow trout farms
in the UK, this means that 40% of farms are still free of the disease. It is possible for them to
remain RMS-free, or even to clear the disease with some effort. Knowing where the threats
are coming from is the key for doing this. By far the highest risk of introducing RMS to a
farm is by moving live fish. This is an essential practice for any farm, but selecting an RMSfree source will limit the risk of infection dramatically. As recent research4 has shown, having
less than four suppliers greatly reduces the risk. The second main route of infection is the
water source. This may seem difficult to control, but having an influence over what is
upstream of the site might help to prevent disease outbreaks. This ties in with the strict
biosecurity that should be maintained on the farm. Recent research has also suggested that
mechanical handling should be kept to a minimum. There are farms that have cleared the
disease and have had no reoccurrence: this was achieved by implementing all of the above
and completely clearing, fallowing and disinfecting ponds.
Treatment of RMS
It is possible to treat fish with RMS. The easiest way is to leave the fish and let the lesions
resolve on their own accord, without any visible scaring occurring. If possible fish can be
hand-graded and fish with lesions selected and left to allow the lesions to heal. It has to be
said that stress has been reported to have different effects on RMS infection: some farmers
have noticed that after stressing the fish the lesions healed up more quickly, whereas other
farmers have found stress aggravates the situation. Another possibility is to fillet the fish, but
that is not always an option. Topical treatments with all sorts of disinfectants have been tried:
results are mixed, but they appear to make the lesions appear less severe rather the resolving
them. As said before, affected fish can be treated with a range of antibiotics, florfenicol and
oxytetracycline being the two most commonly used. However, they should only be used as a
last resort: antibiotic resistance and residues in the flesh are issues not to be taken lightly, and
in any case the disease will resolve itself in time.
Conclusions and Future work
Two bacterial collections were established over the course of the project. From the RMS
sampling, 108 Gram -ve filamentous yellow pigmented bacteria were collected from both
healthy and RMS affected fish during the project. Although the Flavobacterium spp. in this
collection have not been fully speciated as yet, there does not appear to be a correlation
between RMS-affected fish and the level of Flavobacterium sp. found compared with
unaffected fish. The IHC against with F. psychrophilum were mainly negative (with a few
sections having inconclusive staining both from positive and negative farms). Also there was
no difference in the serology between infected and non-infected farms or the isolates used for
this screening. This was carried out to determine if the RMS-affected fish develop antibodies
against F. psychrophilum or/and RLO during an RMS infection.
Characterisation of F. psychrophilum from clinical samples using PFGE analysis was not
completed as it was decided that is was first necessary to complete the speciation of the
Flavobacterim collection and the RMS samples so that the groupings obtained with the PFGE
results would have some significance with respect to different Flavobacterium spp. The
serotyping Western blotting system gave many more profile types than the original four
found when the system was set up. It remains to be established how these relate to different
17
Flavobacterium spp. It was found that the PCR reacts with Flavobacterium sp. closely related
to F. psychrophilum, suggesting that the PCR is in fact not completely specific for F.
psychrophilum. We need to complete the sequencing of the isolates to confirm this.
It was possible to identify an RLO in RMS and SD samples using a qPCR method with
high numbers of positive samples found in affected fish. What was also seen was that the
amount of RLO in the lesion was significantly higher than the other organs or unaffected
skin. However attempts to look for the RLO in formalin-fixed wax-embedded archive
samples were unsuccessful, possibly due to damaged DNA upon its extraction.
No conclusive results were obtained from the cell culture work performed during the
study (in an attempt to isolate the RLO), and although a variety of bacterial isolates were
obtained on PsA, these remain to be identified (collected from local fish farms). Several
swabs from skin lesions and spleen of RMS-postive fish produced very small white colonies,
but it has not been possible to passage these cultures on to fresh medium.
It was not possible to test the efficacy of an existing SRS vaccine against RMS because it
was not a registered vaccine, it would be very useful to do this with a commercially available
vaccine from a different supplier.
In conclusion, although Flavobacterium spp. have been found in fish with RMS, no firm
association can be made between this bacterium and RMS. In addition, although there was a
strong association between presence of the RLO by qPCR and RMS-affected fish samples
this does not prove causality of the disease by the RLO. However, with the number of
samples analysed and the consistent results obtained, it is possible to say that that the RLO
does seem to be involved in the disease.
Despite additional effort in staff time and reagents during this project some of the work
remains to be completed due the large volume of data generated. We would like to request an
extension to the project in order to complete all the analysis and perform the additional tasks
that will inform on management strategies. Our colleagues at Marine Scotland have indicated
that they would be prepared to join the project and assist on some of the tasks. They will
provide the staff time in kind for the project. The suggested objectives and contributors are
listed below:






To compliment and utilise the data derived from serotyping, MALDI BioTyping, and
PFGE we recommend completion of the 16S rDNA sequence analysis of the RMS and
Flavobacterium collections, to allow the final, full and accurate characterisation of the
relevant isolates in these collections.
Prepare subtractive library and non-subtractive library to determine if RLO or viral genes
are present in RMS fish (CEFAS and Stirling) (using next generation sequencing - either
454 or Illumina (subcontracted to either VLA, Fera or Liverpool Uni) to determine which
species are involved and if there is any link between species and occurrence during RMS
Optimise the current qPCR used at CEFAS and transfer the method to both Stirling and
Marine Scotland laboratories (Technology transfer CEFAS, Stirling and Marine Scotland)
Perform qPCR on eggs (Marine Scotland and Stirling).
Host response- which cells and genes are involved? PCR, cell staining, functional assays
(Marine Scotland and Stirling)
Attempt to culture the aetiological agent on cell lines (Stirling and Marine Scotland),
checking on what has been tried already at CEFAS and MS.Vaccination with existing
commercial Rickettsia vaccine (small trial on fish farm) CEFAS, Stirling and Marine
Scotland
18
As mentioned above there has been substantial added to value to this project which we have
summarised below.
Added Value to current RMS project:
(1) Work at CEFAS
Consumables
Various consumables for RLO molecular lab work - tips, tubes for homogenisation, gloves etc - approx £200
Production and cloning of RLO PCR for production of standards for qPCR approx £200
£950 - August 2010 - Mastermix (5x 5ml = 20 plates worth)
£606 - May 2010 DNA extraction plates x2
£350 – Flavo PFGE consumables (enzymes, media, plasticware etc)
(Total £2306.00)
Staff time (man days)
Meetings - 6 days
RLO Lab work preparing standard material -5 days
RLO Lab work supervision – 10 days
Flavo labwork and supervision – 10 days
Statistician – 1 day
Histopathology – 1 day
Epidemiologist – 1 day
(Total £7390.00)
Total £9700
(1) Co-habitation study in USA £2500
10 days +fish +facilities
(2) qPCR analysis by Doug Call’s group £2000
10 days + consumables
(3)Two associated MSc projects £5000
10 days supervision + 3 months/student* + consumables
*Not included in time re final report
(4) MALDI BioTyper™ analysis in Korea £1500
5 days + consumables
(5) BTA – sampling at farm sites (Seven different sites) £3500
5 days
Total estimated added value £24,200
19

Sarf057 Development Of Improved Management Strategies For Red

Transcription

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